Work in our laboratory has been concerned with the regulation of virulence factors in Bordetella pertussis, the causative agent of whooping cough, and the target of pertussis vaccine. Previous studies have shown that a genetic locus, the bvg locus, is responsible for the regulation of B. pertussis virulence determinants (pertussis toxin, filamentous hemagglutinin, etc.; major components of the pertussis vaccine) in response to environmental stimuli. We have recently developed powerful new physical and genetic methods for the analysis of bacterial genomes. We have used these tools in the past year in the analysis of regulation of expression of pertussis toxin, the major protective antigen in pertussis vaccines. Although expression of this gene is governed by the bvg locus, this regulation is not direct and it appears that other factors may be involved. We have conducted a genetic search for such factors and have failed to identify them. However, a very interesting new class of mutations in the bvg locus has been discovered. Mutations in this class affect regulation of pertussis toxin but not some other virulence factors such as filamentous hemagglutinin. We have examined over 100 revertants of these mutations which restore pertussis toxin expression. Some of these reverting mutations map back to the bvg locus, however, others affect the pertussis toxin gene itself. In these cases, pertussis toxin expression is no longer under the control of the bvg locus. These revertant strains have several characteristics that make them potentially exciting candidates for vaccine production: 1) The level of expression of the pertussis toxin gene is, in some revertants, higher than in the parent strain. 2) The pertussis toxin gene is expressed under conditions where the bvg locus shuts off expression of the other virulence factors, thus making it possible to produce pertussis toxin under conditions where other toxic factors are not produced, thus potentially improving vaccine safety as well as safety in production. 3) Expression of pertussis toxin, being no longer under the control of the bvg locus, is not subject to phase-variation which can be a problem in large scale fermentors and result in a very low yields.